1. Field of the Present Disclosure
The present disclosure provides a library of synthetic standard molecules for multiple species of microbial pathogens, including Cryptosporidium, Giardia, and microsporidia. Each of these standard molecules includes a bacterial plasmid molecule containing a specific DNA sequence insert that represents a consensus sequence of the 18s rRNA gene for a single species of interest. These standard molecules may be used by, for example, researchers, utility operators, and clinical laboratory technicians as a surrogate for native genomic DNA in a variety of situations.
2. Related Art
Due to technological limitations, environmental and clinical laboratories are increasingly moving away from microscopic methods and towards molecular detection methods. Molecular methods typically use the polymerase chain reaction (PCR) to detect a specific DNA sequence in the genome of a target organism. Compared to microscopic methods, molecular methods offer increased speed, sensitivity, and reproducibility. Molecular methods can also provide supplementary data unattainable using microscopy, such as, for example, genotype identification.
One microbial pathogen of particular interest is Cryptosporidium. Fifteen waterborne Cryptosporidium outbreaks were reported in the United States between 1991 and 2002, affecting over 408,000 individuals. This makes Cryptosporidium the highest cause of waterborne disease by number of affected individuals. The most significant outbreak occurred in Milwaukee, Wis. in 1993. This well-studied case affected over 403,000 individuals and cost the region an estimated $96.2 million. This event, plus several major recreational outbreaks since then, underscores the importance of proper water monitoring.
Giardia contamination can cause outbreaks that result in similar disruptions.
Microsporidia, including Encephalitozoon intestinalis and Enterocytozoon bieneusi, cause microspridiosis, which is an opportunistic infection that can cause diarrhea and wasting in immunocompromised patients.
Unfortunately, the introduction of new molecular tools for targets such as, e.g., Cryptosporidium and Giardia has been restricted by the lack of standardized positive controls. Positive controls, typically purified genomic DNA from pathogens of interest, may have multiple roles in the development and validation of a molecular method. Two of the most important roles include:                As a sensitivity control, determining detection limits and quantifying target DNA; and        As a specificity control, resolving target genotypes.        
It can be extremely challenging to obtain positive controls for microbial pathogens of environmental interest. Many such organisms are difficult to culture in vitro. The distribution of others is regulated by the Centers for Disease Control and/or the United States Department of Commerce. Researchers who wish to develop new tests for these pathogens must often perform their own isolations from clinical or environmental samples or obtain specimens from collaborators. These research stocks are often subject to inconsistent quality control, increase the risk of laboratory-associated infections, and are of insufficient quantity for industrial-scale development and validation. Until an alternative source can be developed, the limited availability of positive controls threatens to prevent the introduction of any new molecular tests for microbial pathogens into the market.
In the case of Cryptosporidium, limited amounts of positive control DNA are available from a handful of Biological Resource Centers (BRCs). In particular, Waterborne supplies purified C. parvum and C. muris oocysts, while AMERICAN TYPE CULTURE COLLECTION™ (ATCC) can regularly provide researchers with genomic DNA from C. parvum (Iowa strain). A third commercial source, the Biodefense and Emerging Infections Research Resources Repository (BEI Resources), supplies genomic DNA and other reagents only to NIH-funded investigators. These supplies are insufficient for widespread method development, especially for assays aimed at distinguishing multiple genotypes. As a result, test development has been fragmented as research groups rely on various organism stocks of inconsistent quality.
Accordingly, there exists a pressing need for standardized positive controls for Cryptosporidium, Giardia, microsporidia, and other microbial pathogens that may be used to develop and validate moleculr detection and genotyping methods.